Development of high amylose wheat through TILLING

BACKGROUND: Wheat (Triticum spp.) is an important source of food worldwide and the focus of considerable efforts to identify new combinations of genetic diversity for crop improvement. In particular, wheat starch composition is a major target for changes that could benefit human health. Starches with increased levels of amylose are of interest because of the correlation between higher amylose content and elevated levels of resistant starch, which has been shown to have beneficial effects on health for combating obesity and diabetes. TILLING (Targeting Induced Local Lesions in Genomes) is a means to identify novel genetic variation without the need for direct selection of phenotypes. RESULTS: Using TILLING to identify novel genetic variation in each of the A and B genomes in tetraploid durum wheat and the A, B and D genomes in hexaploid bread wheat, we have identified mutations in the form of single nucleotide polymorphisms (SNPs) in starch branching enzyme IIa genes (SBEIIa). Combining these new alleles of SBEIIa through breeding resulted in the development of high amylose durum and bread wheat varieties containing 47-55% amylose and having elevated resistant starch levels compared to wild-type wheat. High amylose lines also had reduced expression of SBEIIa RNA, changes in starch granule morphology and altered starch granule protein profiles as evaluated by mass spectrometry. CONCLUSIONS: We report the use of TILLING to develop new traits in crops with complex genomes without the use of transgenic modifications. Combined mutations in SBEIIa in durum and bread wheat varieties resulted in lines with significantly increased amylose and resistant starch contents

Prospecting for Energy-Rich Renewable Raw Materials: Sorghum Stem Case Study.

Sorghum vegetative tissues are becoming increasingly important for biofuel production. The composition of sorghum stem tissues is influenced by genotype, environment and photoperiod sensitivity, and varies widely between varieties and also between different stem tissues (outer rind vs inner pith). Here, the amount of cellulose, (1,3;1,4)-β-glucan, arabinose and xylose in the stems of twelve diverse sorghum varieties, including four photoperiod-sensitive varieties, was measured. At maturity, most photoperiod-insensitive lines had 1% w/w (1,3;1,4)-β-glucan in stem pith tissue whilst photoperiod-sensitive varieties remained in a vegetative stage and accumulated up to 6% w/w (1,3;1,4)-β-glucan in the same tissue. Three sorghum lines were chosen for further study: a cultivated grain variety (Sorghum bicolor BTx623), a sweet variety (S. bicolor Rio) and a photoperiod-sensitive wild line (S. bicolor ssp. verticilliflorum Arun). The Arun line accumulated 5.5% w/w (1,3;1,4)-β-glucan and had higher SbCslF6 and SbCslH3 transcript levels in pith tissues than did photoperiod-insensitive varieties Rio and BTx623 (<1% w/w pith (1,3;1,4)-β-glucan). To assess the digestibility of the three varieties, stem tissue was treated with either hydrolytic enzymes or dilute acid and the release of fermentable glucose was determined. Despite having the highest lignin content, Arun yielded significantly more glucose than the other varieties, and theoretical calculation of ethanol yields was 10 344 L ha-1 from this sorghum stem tissue. These data indicate that sorghum stem (1,3;1,4)-β-glucan content may have a significant effect on digestibility and bioethanol yields. This information opens new avenues of research to generate sorghum lines optimised for biofuel production

Cell wall characteristics in stems of grain, sweet and wild sorghum lines.

<p>Transmission electron micrographs of transverse sections of pith, rind and epidermal tissues for BTx623, Rio and Arun. Images are all of the same scale, bar indicates 100 μm. Ground tissue (GT), vascular bundle (VB), epidermis (EPI).</p

Distribution of glucan polymers in stems of grain, sweet and wild sorghum lines.

<p>Sections of mature sorghum stem were stained with Calcofluor-white and images captured with a dissection microscope and a fluorescence filter. All pictures are of the same scale, bar is 300 μm. Ground tissue (GT), vascular bundle (VB), epidermis (EPI).</p

Cell wall components in grain, sweet and wild sorghum lines.

<p>(<b>a</b>) Klason lignin, (<b>b</b>) Updegraff cellulose, (<b>c</b>) total arabinose plus xylose, and (<b>d</b>) (1,3;1,4)-β-glucan in sorghum stem sections. Mean and standard error of n = 3 biological replicates, three technical replicates per assay. Different letters indicate statistical differences (ANOVA details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156638#pone.0156638.s003" target="_blank">S3 Table</a>).</p

Diversity in morphological and physiological traits in wild, sweet and grain sorghum lines.

<p>Percentage contribution of grain head, leaf and stem to plant dry weight (mean; with SEM indicated by vertical line; n = 3 biological replicates) in mature (<b>b</b>) grain (<i>S</i>. <i>bicolor</i> ‘BTx623’), (<b>c</b>) sweet (<i>S</i>. <i>bicolor</i> ‘Rio’) and (<b>d</b>) wild (<i>S</i>. <i>bicolor</i> ssp. <i>verticilliflorum</i> ‘Arun’) sorghum lines. Rio and BTx623 plant were photographed 129 d after planting and Arun 159 d after planting.</p

Transcript analysis in sorghum stem sections.

<p>Transcript profile of (a) <i>SbCslH3</i> and (<b>b</b>) <i>SbCslF6</i> in pith, rind and whole stem tissues of Rio and Arun. The transcript levels of these genes in the stem of mature BTx623 were reported previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156638#pone.0156638.ref038" target="_blank">38</a>]. Mean and standard errors n = 3 biological replicates. Data are normalised to control genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156638#pone.0156638.s004" target="_blank">S4 Table</a>).</p